Compositions and methods for assessing the risk of cancer occurrence

ABSTRACT

The present invention provides a method for evaluating the risk of occurrence of cancer in an individual.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage Application pursuant to 35U.S.C. § 371 of International Patent Application PCT/EP2017/050034,filed on Jan. 2, 2017, and published as WO 2017/114973 on Jul. 6, 2017,which claims priority to European Patent Application 16305133.7, filedon Feb. 5, 2016, and European Patent Application 15307190.7, filed onDec. 31, 2015, all of which are incorporated herein by reference intheir entireties for all purposes.

INTRODUCTION

Cancer is a multi-faceted disease in which a group of cells displayuncontrolled growth, invasion that intrudes upon and destroys adjacenttissues, and sometimes metastasis, or spreading to other locations inthe body via lymph or blood. These three malignant properties of cancersdifferentiate them from benign tumors, which do not invade ormetastasize.

There are a number of methods currently used to treat each type ofcancer, including surgery, radiotherapy, chemotherapy and targetedtherapy. Successful cancer therapy is directed to the primary tumor andto any metastases, whether clinically apparent or microscopic.

The selection of an appropriate treatment is crucial for the patient. Itis essential to know when to use immediately a heavy and aggressivetreatment protocol in order to prevent extension of an aggressivecancer. In contrast, performing a heavy and aggressive treatment when itis not necessitated by the tumor carried by the patient is alsodisadvantageous for the patient. Indeed, heavy and aggressive treatmentsalways lead to adverse toxicities that may significantly affect thepatient's quality of life. For example most of them are mutagenic andare thus prone to induce secondary tumors. In addition, such heavy andaggressive treatments are usually very costly, and should thus beperformed only when it is necessary.

Currently, treatment selection for solid tumors is based on tumorstaging, which is usually performed using the Tumor/Node/Metastasis(TNM) test from the American Joint Committee on Cancer (AJCC). The TNMsystem assigns a number based on three categories. “T” denotes the tumorsize, “N” the degree of lymphatic node involvement, and “M” the degreeof metastasis. The broader stage of a cancer is usually quoted as anumber I, II, III, IV derived from the TNM value grouped by prognosis; ahigher number indicates a more advanced cancer and likely a worseoutcome.

It is commonly acknowledged that, while this test and staging systemprovides some valuable information concerning the stage at which solidcancer has been diagnosed in the patient, it is imprecise andinsufficient. In particular, it is limited to solid tumors. Liquidtumors on the other hand are mostly characterized by the identificationof cytogenetic alterations.

Most importantly, the TNM test fails to identify the earliest stages oftumor progression. These early stages offer the most promising windowfor therapy. Detection of a cancer at the very beginning of itsdevelopment allows targeted, efficient therapy, with reducedside-effects. It is thus important to identify patients at the earliestpossible stage as a part of a screening of the whole population. Cancercan thus be identified in a community early, enabling earlierintervention and management to reduce mortality and suffering from saiddisease. There is a real need for better prognosis tests of theoccurrence of cancer, not only to improve patient global survival, butalso to improve their quality of life and to keep aggressive and costlychemotherapies for patients who will really benefit from them. Inparticular, there is a need for a test assessing the risk of a subjectto develop a cancer.

DESCRIPTION

The present invention provides a simple and efficient method forprognosing the occurrence of a cancer in subjects who have never beenpreviously diagnosed with cancer. The present inventors have shown thatthe presence of progastrin in sample of a subject is a good and reliableindication of whether said subject will develop cancer. This relation isindependent of age or any other parameter. Progastrin thus offers asimple and efficient tool for determining the risks of a subject ofdeveloping a cancer. Progastrin is thus a marker of the earliest stagesof cancer.

Prognosis tests based on progastrin levels have been describedpreviously. However, such tests are limited to predicting the risk of apatient having hyperplastic polyp, of developing a colonic neoplasiaafter resection of said hyperplastic polyps (WO 2012/164035; Do et al.,Cancer Prev Res, 5(4): 675-684, 2012). Such tests are thus of a limiteduse, since they are restricted to prognosing a cancer which is alreadyknown to be present. They cannot be used to evaluate the risk of asubject who does not have any sign of cancer of developing this disease.

In a first aspect, the invention relates to a method of evaluating therisk of the occurrence of a cancer in a subject who has not beenpreviously diagnosed with cancer, said method comprising steps of:

-   -   a) determining the level of progastrin in sample of said        subject;    -   b) determining the risk that said subject will develop a cancer        based on the level of step a).

A ROC (Receiver Operating Characteristic) analysis of clinical data hasshown that the presence of progastrin is a highly specific and sensitivemarker. Progastrin is essentially not detectable in samples of subjectswho do not develop cancer. On the other hand, a subject who has neverbeen diagnosed with cancer has a non-negligible risk of developing acancer in the future if progastrin is detectable in his/her sample.

The method of the invention makes it possible to evaluate easily andreliably the risk of a subject who has not been previously diagnosedwith cancer to develop cancer. In other words, this method also enablesthe identification of subjects who will develop cancer, even though theypresently display no symptom. Such patients already have cancer, eventhough they perceive no symptom thereof.

The expression “evaluation of a risk of development of a cancer in asubject” designates the determination of a relative probability for agiven subject to display symptoms of cancer in the future. A methodaccording to the invention represents a tool in the evaluation of saidrisk, in combination with other methods or indicators such as clinicalexamination, biopsy and determination of the level of a known biomarkerof cancer, such as, for example, CA125 and/or OVA1.

A “subject” which may be subjected to the methodology described hereinmay be any of mammalian animals including human, dog, cat, cattle, goat,pig, swine, sheep and monkey. A human subject can be known as a“patient”. Preferably, a ““subject” is a mammal that is not sufferingfrom cancer and is not suspected of suffering from cancer and has notbeen diagnosed with cancer. As used herein, a “subject suffering fromcancer” refers to a mammal that is suffering from cancer and showssymptoms thereof, or has been diagnosed with cancer. A subject has been“diagnosed with cancer” when a medical test conducted by a medicalpractitioner has revealed the presence of cancer. As used herein, a“symptom” is any subjective evidence of disease, e.g., cancer. A“symptom” is a departure from normal function or feeling which isnoticed by a patient, reflecting the presence of an unusual state, or ofa disease, e.g., cancer.

A disease is considered asymptomatic if a patient is a carrier for saiddisease, but experiences no symptom. Asymptomatic conditions may not bediscovered until the patient undergoes medical tests, such as, e.g.,measuring the progastrin level.

The present method is also particularly useful because it allows one toidentify a cancer in a subject, even when the subject has never beendiagnosed with cancer and/or does not experience any symptom thereof.Progastrin is a highly specific and sensitive cancer marker. Thedetection of progastrin in a subject indicates that there is a highlikelihood that said subject will develop a cancer. Progastrin is thus aparticularly important biomarker for identifying subjects who willdevelop cancer, even though they do not display any symptoms yet. Theinvention is particularly advantageous because it allows screening apopulation of subjects seemingly healthy, i.e., who have never beendiagnosed with cancer and/or have not experienced any symptom thereof,and identifying those who will develop cancer. By “screening” it isherein referred to a method used to identify within a population thepossible presence of an as-yet-undiagnosed disease in individualswithout signs or symptoms. This can include individuals withpre-symptomatic or unrecognized symptomatic disease. It will be clear tothe skilled person that as such, screening tests are somewhat unique inthat they are performed on persons apparently in good health. Theproximate goal of cancer screening is the identification of early stagecancer, or precancerous lesions, before a person develops symptoms andat a point in the disease trajectory when treatment is likely to resultin cure.

In another aspect, the invention provides a method of prognosing acancer in a subject who has not been previously diagnosed with cancer,said method comprising the steps of:

-   -   a) determining the level of progastrin in sample of said        subject;    -   b) prognosing a cancer based on the level of step a).

“Prognosis” as used herein means the likelihood of recovery from adisease or the prediction of the probable development or outcome of adisease. For example, if a sample from a subject is negative for thepresence of progastrin, then the “prognosis” for that subject is betterthan if the sample is positive for progastrin.

By “progastrin”, it is herein referred to the mammalian progastrinpeptide. Progastrin is formed by cleavage of the first 21 amino acids(the signal peptide) from preprogastrin, a 101 amino acids peptide(Amino acid sequence reference: AAB19304.1) which is the primarytranslation product of the gastrin gene. The 80 amino acid chain ofprogastrin is further processed by cleavage and modifying enzymes toseveral biologically active gastrin hormone forms: gastrin 34 (G34) andglycine-extended gastrin 34 (G34-Gly), comprising amino acids 38-71 ofprogastrin, gastrin 17 (G17) and glycine-extended gastrin 17 (G17-Gly),comprising amino acids 55 to 71 of progastrin.

In a preferred embodiment, the progastrin peptide of the invention ishuman progastrin. More preferably, the expression “human progastrin”refers to the human PG of sequence SEQ ID No. 1. Human progastrincomprises notably a N-terminus and a C-terminus domains which are notpresent in the biologically active gastrin hormone forms mentionedabove. Preferably, the sequence of said N-terminus domain is representedby SEQ ID NO. 2. In another preferred embodiment, the sequence of saidC-terminus domain is represented by SEQ ID NO. 3.

The present invention provides methods for detection of progastrin insamples, especially of biological samples such as biological fluids andcells, tissues, biopsy samples and organ sections etc.

By “biological sample” it is herein referred to any sample that may betaken from a subject. Such a sample must allow for the determination ofthe expression levels of progastrin. Progastrin is known to be asecreted protein. Preferred biological samples for the determination ofthe level of the progastrin protein thus include biological fluids. A“biological fluid” as used herein means any fluid that includes materialof biological origin. Preferred biological fluids for use in the presentinvention include bodily fluids of an animal, e.g. a mammal, preferablya human subject. The bodily fluid may be any bodily fluid, including butnot limited to blood, plasma, serum, lymph, cerebrospinal fluid (CSF),saliva, sweat and urine. Preferably, said preferred liquid biologicalsamples include samples such as a blood sample, a plasma sample, or alymph sample. More preferably, the biological sample is a blood sample.Indeed, such a blood sample may be obtained by a completely harmlessblood collection from the patient and thus allows for a non-invasiveassessment of the risks that the subject will develop a tumor.

A “biological sample” as used herein also includes a solid cancer sampleof the patient to be tested, when the cancer is a solid cancer. Suchsolid cancer sample allows the skilled person to perform any type ofmeasurement of the level of the biomarker of the invention. In somecases, the methods according to the invention may further comprise apreliminary step of taking a solid cancer sample from the patient. By a“solid cancer sample”, it is referred to a tumor tissue sample. Even ina cancerous patient, the tissue which is the site of the tumor stillcomprises non tumor healthy tissue. The “cancer sample” should thus belimited to tumor tissue taken from the patient. Said “cancer sample” maybe a biopsy sample or a sample taken from a surgical resection therapy.

According to one aspect, the sample from the patient is a cancer cell ora cancer tissue.

This sample may be taken and if necessary prepared according to methodsknown to a person skilled in the art. In particular, it is well known inthe art that the sample should be taken from a fasting subject.

The cancer cell or cancer tissue in the present invention is notparticularly limited.

As used herein, the term “cancer” refers to or describes thephysiological condition in mammals that is typically characterized byunregulated cell proliferation. The terms “cancer” and “cancerous” asused herein are meant to encompass all stages of the disease. A “cancer”as used herein is any malignant neoplasm resulting from the undesiredgrowth, the invasion, and under certain conditions metastasis ofimpaired cells in an organism. The cells giving rise to cancer aregenetically impaired and have usually lost their ability to control celldivision, cell migration behavior, differentiation status and/or celldeath machinery. Most cancers form a tumor but some hematopoieticcancers, such as leukemia, do not.

Thus, a “cancer” as used herein may include both benign and malignantcancers. Examples of cancer include but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukaemia or lymphoid malignancies.More specifically, a cancer according to the present invention isselected from the group comprising squamous cell cancer (e.g.,epithelial squamous cell cancer), lung cancer including small-cell lungcancer, non-small cell lung cancer, adenocarcinoma of the lung andsquamous carcinoma of the lung, oropharyngeal cancer, nasopharyngealcancer, laryngeal cancer, cancer of the peritoneum, oesophageal cancer,hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer and gastrointestinal stromal cancer, pancreaticcancer, glioblastoma, brain cancer, nervous system cancer, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, cancer of theurinary tract, hepatoma, breast cancer, colon cancer, rectal cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney or renal cancer, prostate cancer, gallbladder cancer,vulval cancer, testicular cancer, thyroid cancer, Kaposi sarcoma,hepatic carcinoma, anal carcinoma, penile carcinoma, non-melanoma skincancer, melanoma, skin melanoma, superficial spreading melanoma, lentigomaligna melanoma, acral lentiginous melanomas, nodular melanomas,multiple myeloma and B-cell lymphoma (including Hodgkin lymphoma;non-Hodgkin lymphoma, such as e.g., low grade/follicular non-Hodgkin'slymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chroniclymphocytic leukaemia (CLL); acute lymphoblastic leukaemia (ALL); hairycell leukaemia; chronic myeloblastic leukaemia (CML); Acute MyeloblasticLeukaemia (AML); and post-transplant lymphoproliferative disorder(PTLD), as well as abnormal vascular proliferation associated withphakomatoses, oedema (such as that associated with brain tumors), Meigs'syndrome, brain, as well as head and neck cancer, including lip & oralcavity cancer, and associated metastases.

In a preferred embodiment, said cancer is lung cancer, lip & oral cavitycancer, oropharyngeal cancer, nasopharyngeal cancer, laryngeal cancer,prostate cancer, oesophageal cancer, gallbladder cancer, liver cancer,hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer and gastrointestinal stromal cancer, pancreaticcancer, Hodgkin lymphoma, Non-Hodgkin lymphoma, leukemia, multiplemyeloma, Kaposi sarcoma, kidney cancer, bladder cancer, colon cancer,rectal cancer, colorectal cancer, hepatoma, hepatic carcinoma, analcarcinoma, thyroid cancer, non-melanoma skin cancer, skin melanoma,brain cancer, nervous system cancer, testicular cancer, cervical cancer,uterine cancer, endometrial cancer, ovarian cancer, or breast cancer.

In a more preferred embodiment, said cancer is oesophageal cancer, livercancer, hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer and gastrointestinal stromal cancer, pancreaticcancer, Hodgkin lymphoma, colon cancer, rectal cancer, colorectalcancer, hepatoma, hepatic carcinoma, anal carcinoma, non-melanoma skincancer, skin melanoma, cervical cancer, uterine cancer, endometrialcancer, ovarian cancer, or breast cancer.

Preferably, the risk that said subject will develop a cancer isdetermined in step b) by comparing the level of step a) with a referencelevel.

The term “reference level”, as used herein, refers to the expressionlevel of the cancer marker under consideration, i.e. progastrin, in areference sample. A “reference sample”, as used herein, means a sampleobtained from subjects, preferably two or more subjects, known to befree of the disease or, alternatively, from the general population. Thesuitable reference expression levels of the cancer marker can bedetermined by measuring the expression levels of said cancer marker inseveral suitable subjects, and such reference levels can be adjusted tospecific subject populations. The reference value or reference level canbe an absolute value; a relative value; a value that has an upper or alower limit; a range of values; an average value; a median value, a meanvalue, or a value as compared to a particular control or baseline value.A reference value can be based on an individual sample value such as,for example, a value obtained from a sample from the subject beingtested, but at an earlier point in time. The reference value can bebased on a large number of samples, such as from population of subjectsof the chronological age matched group, or based on a pool of samplesincluding or excluding the sample to be tested.

Advantageously, a “reference level” is a predetermined progastrin level,obtained from a biological sample from a subject with a known particularstatus as regards cancer. In particular embodiments, the reference levelused for comparison with the test sample in step (b) may have beenobtained from a biological sample from a healthy subject, or from abiological sample from a subject suffering from cancer; it is understoodthat the reference expression profile can also be obtained from a poolof biological samples of healthy subjects or from a pool of samples fromsubjects having cancer. The present inventors have shown that the levelof progastrin in fasting, healthy subjects is 0 pM. In a preferredembodiment, the reference level is 0 pM.

The levels of progastrin can be measured by any method known to theperson of skill in the art.

Preferably, determining the levels of progastrin in a sample includescontacting said sample with a progastrin-binding molecule and measuringthe binding of said progastrin-binding molecule to progastrin.

When expression levels are measured at the protein level, it may benotably performed using specific progastrin-binding molecules, such ase.g., antibodies, in particular using well known technologies such ascell membrane staining using biotinylation or other equivalenttechniques followed by immunoprecipitation with specific antibodies,western blot, ELISA or ELISPOT, enzyme-linked immunosorbant assays(ELISA), radioimmunoassays (RIA), immunohistochemistry (IHC),immunofluorescence (IF), antibodies microarrays, or tissue microarrayscoupled to immunohistochemistry. Other suitable techniques include FRETor BRET, single cell microscopic or histochemistry methods using singleor multiple excitation wavelength and applying any of the adaptedoptical methods, such as electrochemical methods (voltametry andamperometry techniques), atomic force microscopy, and radio frequencymethods, e.g. multipolar resonance spectroscopy, confocal andnon-confocal, detection of fluorescence, luminescence,chemiluminescence, absorbance, reflectance, transmittance, andbirefringence or refractive index (e.g., surface plasmon resonance,ellipsometry, a resonant mirror method, a grating coupler waveguidemethod or interferometry), cell ELISA, flow cytometry, radioisotopic,magnetic resonance imaging, analysis by polyacrylamide gelelectrophoresis (SDS-PAGE); HPLC-Mass Spectroscopy; LiquidChromatography/Mass Spectrometry/Mass Spectrometry (LC-MS/MS)). Allthese techniques are well known in the art and need not be furtherdetailed here. These different techniques can be used to measure theprogastrin levels.

The progastrin-binding molecules of the present invention, especiallythe anti-progastrin antibodies, are particularly useful in animmunoassay. The immunoassay may be an enzyme-linked immunosorbent assay(ELISA), a radioimmunoassay (RIA), an immunodiffusion assay, or animmuno-detection assay, such as a surface plasmon resistance assay (e.g.a Biacore® assay), an ELISPOT, slot-blot, or a western blot. As ageneral guide to such techniques, see for instance, Ausubel et al. (eds)(1987) in “Current Protocols in Molecular Biology” John Wiley and Sons,New York, N.Y.

Antibodies are key reagents in numerous assay techniques used inmedical, veterinary and other immunodetection fields. Such tests includemany routinely used immunoassay techniques, such as for example,enzyme-linked ELISA, RIA, IHC, and IF assays. The level of progastrin ispreferentially assayed by any method known to one of skill in the artusing antibodies directed against said protein. Preferably, the level ofprogastrin is determined using an immunoenzymatic assay, preferablybased on techniques chosen between RIA and ELISA, with at least oneprogastrin-binding molecule. Most preferably, said level is determinedby ELISA with at least one progastrin-binding molecule. More preferably,the level of progastrin is measured with one progastrin-bindingmolecule, using an immunoenzymatic assay, most preferably an ELISAassay.

In a particularly useful embodiment, the method for evaluating the riskof the occurrence of a cancer according to the invention comprisesdetermining the level of progastrin in a biological sample from asubject using an immunoenzymatic assay, preferably based on techniqueschosen between RIA and ELISA, with a progastrin-binding molecule.

These techniques are particularly useful, in that they allow the skilledperson to assay the presence of progastrin by a simple, reproducible andreliable test. The method of the prior art relied on a semi-quantitativetest, i.e., IHC staining of epithelial cells in the whole polyp. Such amethod is somewhat unreliable. In particular, because of the degree ofsubjectivity associated with the assay, it is difficult to compareresults obtained by different pathologists. In contrast, the method ofthe invention is fully quantitative, objective and highly sensitive.

In another particularly useful embodiment, the method according to theinvention comprises determining the level of progastrin in a biologicalsample from a subject using an immunoenzymatic assay, preferably basedon techniques chosen between RIA and ELISA, with a progastrin-bindingmolecule.

Thus the level of progastrin is determined in step a) of the presentmethod by determining the amount of progastrin which is bound by aprogastrin-binding molecule, preferably by an antibody recognisingprogastrin.

By “progastrin-binding molecule”, it is herein referred to any moleculethat binds progastrin, but does not bind gastrin-17 (G17), gastrin-34(G34), glycine-extended gastrin-17 (G17-Gly), or glycine-extendedgastrin-34 (G34-Gly). The progastrin-binding molecule of the presentinvention may be any progastrin-binding molecule, such as, for instance,an antibody molecule or a receptor molecule. Preferably, theprogastrin-binding molecule is an anti-progastrin antibody or anantigen-binding fragment thereof.

By “binding”, “binds”, or the like, it is herein meant that theantibody, or antigen-binding fragment thereof, forms a complex with anantigen which, under physiologic conditions, is relatively stable.Methods for determining whether two molecules bind one another are wellknown in the art and include, for example, equilibrium dialysis, surfaceplasmon resonance, and the like. In a particular embodiment, saidantibody, or antigen-binding fragment thereof, binds to progastrin withan affinity that is at least two-fold greater than its affinity forbinding to a non specific molecule such as BSA or casein. In a moreparticular embodiment, said antibody, or antigen-binding fragmentthereof, binds only to progastrin.

In a particular embodiment, a biological sample from the subject iscontacted with at least one molecule binding to progastrin, wherein theaffinity of said agent for progastrin is of at least 100 nM, at least 90nM, at least 80 nM, at least 70 nM, at least 60 nM, at least 50 nM, atleast 40 nM, at least 30 nM, at least 20 nM, at least 10 nM, at least 5nM, at least 1 nM, at least 100 pM, at least 10 pM, or at least 1 pM, asdetermined by a method such as above-described.

In a particular embodiment, the present invention relates to a methodfor evaluating the risk of the occurrence of a cancer in a subject whohas not been previously diagnosed with cancer, comprising the detectionof the concentration of progastrin in a biological sample from a subjectwho has not been diagnosed with cancer, wherein said biological sampleis contacted with an anti-hPG antibody, or an antigen-binding fragmentthereof.

In a particular embodiment, the present invention relates to a methodfor the prognosis of cancer, comprising the detection of theconcentration of progastrin in a biological sample from a subject whohas not been diagnosed with cancer, wherein said biological sample iscontacted with an anti-hPG antibody, or an antigen-binding fragmentthereof.

The term “antibody” as used herein is intended to include polyclonal andmonoclonal antibodies. An antibody (or “immunoglobulin”) consists of aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds. Each heavy chain comprises aheavy chain variable region (or domain) (abbreviated herein as HCVR orVH) and a heavy chain constant region. The heavy chain constant regioncomprises three domains, CH1, CH2 and CH3. Each light chain comprises alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region comprisesone domain, CL. The VH and VL regions can be further subdivided intoregions of hypervariability, termed “complementarity determiningregions” (CDR) or “hypervariable regions”, which are primarilyresponsible for binding an epitope of an antigen, and which areinterspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavyand light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g. effector cells) and the first component(Clq) of the classical complement system. Antibodies can be of differentisotypes (namely IgA, IgD, IgE, IgG or IgM).

In a more particular embodiment, said biological sample is contactedwith a progastrin-binding antibody, or an antigen-binding fragmentthereof, selected from the group consisting of: polyclonal antibodies,monoclonal antibodies, chimeric antibodies, humanized antibodies, singlechain antibodies, camelized antibodies, IgA1 antibodies, IgA2antibodies, IgD antibodies, IgE antibodies, IgG1 antibodies, IgG2antibodies, IgG3 antibodies, IgG4 antibodies and IgM antibodies.

In addition, a person skilled in the art of generating antibodies willeasily select and implement a method for generating polyclonal and/ormonoclonal antibodies against a given antigen. Also, a person skilled inthe art knows a method to determine the CDRs within light and heavychains of an antibody.

A “polyclonal antibody” is an antibody which was produced among or inthe presence of one or more other, non-identical antibodies. In general,polyclonal antibodies are produced from a B-lymphocyte in the presenceof several other B-lymphocytes producing non-identical antibodies.Usually, polyclonal antibodies are obtained directly from an immunizedanimal.

The term “monoclonal antibody” designates an antibody arising from anearly homogeneous antibody population, wherein population comprisesidentical antibodies except for a few possible naturally-occurringmutations which can be found in minimal proportions. A monoclonalantibody arises from the growth of a single cell clone, such as ahybridoma, and is characterized by heavy chains of one class andsubclass, and light chains of one type. Anti-human progastrin (anti-hPG)monoclonal antibodies and their use for diagnosis or therapy are alreadyknown in the art, see e.g., WO 2011/083 088 for colorectal cancer, WO2011/083 090 for breast cancer, WO 2011/083 091 for pancreatic cancer,WO 2011/116 954 for colorectal and gastrointestinal cancer, and WO2012/013 609 and WO 2011/083089 for liver pathologies.

By the expression “antigen-binding fragment” of an antibody, it isintended to indicate any peptide, polypeptide, or protein retaining theability to bind to the target (also generally referred to as antigen) ofthe said antibody, generally the same epitope, and comprising an aminoacid sequence of at least 5 contiguous amino acid residues, at least 10contiguous amino acid residues, at least 15 contiguous amino acidresidues, at least 20 contiguous amino acid residues, at least 25contiguous amino acid residues, at least 40 contiguous amino acidresidues, at least 50 contiguous amino acid residues, at least 60contiguous amino residues, at least 70 contiguous amino acid residues,at least 80 contiguous amino acid residues, at least 90 contiguous aminoacid residues, at least 100 contiguous amino acid residues, at least 125contiguous amino acid residues, at least 150 contiguous amino acidresidues, at least 175 contiguous amino acid residues, or at least 200contiguous amino acid residues, of the amino acid sequence of theantibody.

In a particular embodiment, the said antigen-binding fragment comprisesat least one CDR of the antibody from which it is derived. Still in apreferred embodiment, the said antigen binding fragment comprises 2, 3,4 or 5 CDRs, more preferably the 6 CDRs of the antibody from which it isderived.

The “antigen-binding fragments” can be selected, without limitation, inthe group consisting of Fv, scFv (sc for single chain), Fab, F(ab′)₂,Fab′, scFv-Fc fragments or diabodies, or fusion proteins with disorderedpeptides such as XTEN (extended recombinant polypeptide) or PAS motifs,or any fragment of which the half-life time would be increased bychemical modification, such as the addition of poly(alkylene) glycolsuch as poly(ethylene) glycol (“PEGylation”) (pegylated fragments calledFv-PEG, scFv-PEG, Fab-PEG, F(ab′)₂-PEG or Fab′-PEG) (“PEG” forPoly(Ethylene) Glycol), or by incorporation in a liposome, saidfragments having at least one of the characteristic CDRs of the antibodyaccording to the invention. Preferably, said “antigen-binding fragments”will be constituted or will comprise a partial sequence of the heavy orlight variable chain of the antibody from which they are derived, saidpartial sequence being sufficient to retain the same specificity ofbinding as the antibody from which it is descended and a sufficientaffinity, preferably at least equal to 1/100, in a more preferred mannerto at least 1/10, of the affinity of the antibody from which it isdescended, with respect to the target.

In another particular embodiment, a biological sample from a subject iscontacted with least one antibody binding to progastrin, wherein saidantibody has been obtained by an immunization method known by a personskilled in the art, wherein using as an immunogen a peptide which aminoacid sequence comprises the totality or a part of the amino-acidsequence of progastrin. Said antibody may be either polyclonal ormonoclonal. When more than one antibodies are used (e.g., 2), the methodof the invention can be carried out with either only antibodies of thesame type (e.g., two monoclonal antibodies) or antibodies belonging todifferent types (e.g., one monoclonal ad one polyclonal).

In another particular embodiment, said biological sample is contactedwith one such antibody. More particularly, said immunogen comprises apeptide chosen among:

-   -   a peptide which amino acid sequence comprises, or consists of,        the amino acid sequence of full length progastrin, and        particularly full length human progastrin of SEQ ID N^(o) 1,    -   a peptide which amino acid sequence corresponds to a part of the        amino acid sequence of progastrin, and particularly full length        human progastrin of SEQ ID N^(o) 1,    -   a peptide which amino acid sequence corresponds to a part or to        the whole amino acid sequence of the N-terminal part of        progastrin, and in particular peptides comprising, or consisting        of, the amino acid sequence: SWKPRSQQPDAPLG (SEQ ID N^(o) 2),        and    -   a peptide which amino acid sequence corresponds to a part or to        the whole amino acid sequence of the C-terminal part of        progastrin, and in particular peptides comprising, or consisting        of, the amino acid sequence: QGPWLEEEEEAYGWMDFGRRSAEDEN (SEQ ID        N^(o) 3),    -   a peptide which amino acid sequence corresponds to a part of the        amino acid sequence of the C-terminal part of progastrin, and in        particular peptides comprising the amino acid sequence        FGRRSAEDEN (SEQ ID N^(o) 40) corresponding to amino acids 71-80        of progastrin

The skilled person will easily realize that such immunization may beused to generate either polyclonal or monoclonal antibodies, as desired.Methods for obtaining each of these types of antibodies are well knownin the art.

Examples of monoclonal antibodies which were generated by using animmunogen comprising the amino-acid sequence “SWKPRSQQPDAPLG” (SEQ IDN^(o) 2), corresponding to the amino acid sequence 1-14 of humanprogastrin (N-terminal extremity) include, but are not restricted to,monoclonal antibodies designated as: mAb3, mAb4, mAb16, and mAb19 andmAb20, as described in the following Table 1 to Table 4. Othermonoclonal antibodies have been described, although it is not clearwhether these antibodies actually bind progastrin (WO 2006/032980).Experimental results of epitope mapping show that mAb3, mAb4, mAb16, andmAb19 and mAb20 do specifically bind an epitope within said hPGN-terminal amino acid sequence. Polyclonal antibodies recognizingspecifically an epitope within the N-terminus of progastrin representedby SEQ ID NO. 2, have been described in the art (see e.g. WO2011/083088).

TABLE 1 Hybridoma Amino acid deposit mAb sequences SEQ ID NO 6B5B11C10mAb3 VH CDR 1 GYIFTSYW SEQ ID NO 4 VH CDR 2 FYPGNSDS SEQ ID NO 5VH CDR 3 TRRDSPQY SEQ ID NO 6 VL CDR 1 QSIVHSNGNTY SEQ ID NO 7 VL CDR 2KVS SEQ ID NO 8 VL CDR 3 FQGSHVPFT SEQ ID NO 9

TABLE 2 Hybridoma Amino acid deposit mAb sequences SEQ ID NO 20D2C3G2mAb4 VH CDR 1 GYTFSSW SEQ ID NO 10 VH CDR 2 FLPGSGST SEQ ID NO 11VH CDR 3 ATDGNYDWFAY SEQ ID NO 12 VL CDR 1 QSLVHSSGVTY SEQ ID NO 13VL CDR 2 KVS SEQ ID NO 14 VL CDR 3 SQSTHVPPT SEQ ID NO 15

TABLE 3 Hybridoma Amino acid deposit mAb sequences SEQ ID NO 1E9D9B6mAb16 VH CDR 1 GYTFTSYY SEQ ID NO 16 VH CDR 2 INPSNGGT SEQ ID NO 17VH CDR 3 TRGGYYPFDY SEQ ID NO 18 VL CDR 1 QSLLDSDGKTY SEQ ID NO 19VL CDR 2 LYS SEQ ID NO 20 VL CDR 3 WQGTHSPYT SEQ ID NO 21

TABLE 4 Hybridoma Amino acid deposit mAb sequences SEQ ID NO 1B3B4F11mAb19 VH CDR 1 GYSITSDYA SEQ ID NO 22 VH CDR 2 ISFSGYT SEQ ID NO 23VH CDR 3 AREVNYGDSYHFDY SEQ ID NO 24 VL CDR 1 SQHRTYT SEQ ID NO 25VL CDR 2 VKKDGSH SEQ ID NO 26 VL CDR 3 GVGDAIKGQSVFV SEQ ID NO 27

Examples of monoclonal antibodies that can be generated by using animmunogen comprising the amino-acid sequence“QGPWLEEEEEAYGWMDFGRRSAEDEN” (SEQ ID N^(o) 3), (C-terminal part ofprogastrin) corresponding to the amino acid sequence 55-80 of humanprogastrin include, but are not restricted to antibodies designated as:mAb8 and mAb13 in the following Table 5 and 6. Experimental results ofepitope mapping show that mAb13 do specifically bind an epitope withinsaid hPG C-terminal amino acid sequence.

TABLE 5 Hybridoma Amino acid deposit mAb sequences SEQ ID NO 1C10D3B9mAb8 VH CDR 1 GFTFTTYA SEQ ID NO 28 VH CDR 2 ISSGGTYT SEQ ID NO 29VH CDR 3 ATQGNYSLDF SEQ ID NO 30 VL CDR 1 KSLRHTKGITF SEQ ID NO 31VL CDR 2 QMS SEQ ID NO 32 VL CDR 3 AQNLELPLT SEQ ID NO 33

TABLE 6 Hybridoma Amino acid  deposit mAb sequences SEQ ID NO 2C6C3C7mAb13 VH CDR 1 GFIFSSYG SEQ ID NO 34 VH CDR 2 INTFGDRT SEQ ID NO 35VH CDR 3 ARGTGTY SEQ ID NO 36 VL CDR 1 QSLLDSDGKTY SEQ ID NO 37 VL CDR 2LYS SEQ ID NO 38 VL CDR 3 WQGTHFPQT SEQ ID NO 39

Other examples include anti-hPG monoclonal and/or polyclonal antibodiesgenerated by using an immunogen comprising an amino acid sequence of SEQID N^(o) 40.

In a more particular embodiment, in a method according to the inventionsaid biological sample is contacted with at least one anti-hPG antibodyor antigen-binding fragment thereof, preferably with one anti-hPGantibody or antigen-binding fragment thereof, wherein said anti-hPGantibody is chosen among N-terminal anti-hPG antibodies and C-terminalanti-hPG antibodies.

The terms “N-terminal anti-hPG antibodies” and “C-terminal anti-hPGantibodies” designate antibodies binding to an epitope comprising aminoacids located in the N-terminal part of hPG or to an epitope comprisingamino acids located in the C-terminal part of hPG, respectively.Preferably, the term “N-terminal anti-hPG antibodies” refers toantibodies binding to an epitope located in a domain of progastrin whosesequence is represented by SEQ ID NO. 2. In another preferredembodiment, the term “C-terminal anti-hPG antibodies” refers toantibodies binding to an epitope located in a domain of progastrin whosesequence is represented by SEQ ID NO. 3.

The term “epitope” is a region of an antigen that is bound by anantibody. Epitopes may be defined as structural or functional.Functional epitopes are generally a subset of the structural epitopesand have those amino acids that directly contribute to the affinity ofthe interaction. Epitopes may also be conformational. In certainembodiments, epitopes may include determinants that are chemicallyactive surface groupings of molecules such as amino acids, sugar sidechains, phosphoryl groups, or sulfonyl groups, and, in certainembodiments, may have specific three-dimensional structuralcharacteristics, and/or specific charge characteristics. Thedetermination of the epitope bound by an antibody may be performed byany epitope mapping technique, known by a person skilled in the art. Anepitope may comprise different amino acids which located sequentiallywithin the amino acid sequence of a protein. An epitope may alsocomprise amino acids which are not located sequentially within the aminoacid sequence of a protein.

In a particular embodiment of the method of the invention, said antibodyis a monoclonal antibody chosen in the group consisting of:

-   -   A monoclonal antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially three, of        CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID N^(o)        4, 5 and 6, respectively, or sequences with at least 80%,        preferably 85%, 90%, 95% and 98% identity after optimal        alignment with sequences SEQ ID N^(o) 4, 5 and 6, respectively,        and a light chain comprising at least one, preferentially at        least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of        amino acid sequences SEQ ID N^(o) 7, 8 and 9, respectively, or        sequences with at least 80%, preferably 85%, 90%, 95% and 98%        identity after optimal alignment with sequences SEQ ID N^(o) 7,        8 and 9, respectively,    -   A monoclonal antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially three, of        CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID N^(o)        10, 11 and 12, respectively, or sequences with at least 80%,        preferably 85%, 90%, 95% and 98% identity after optimal        alignment with sequences SEQ ID N^(o) 10, 11 and 12,        respectively, and a light chain comprising at least one,        preferentially at least two, preferentially three, of CDR-L1,        CDR-L2 and CDR-L3 of amino acid sequences SEQ ID N^(o) 13, 14        and 15, respectively, or sequences with at least 80%, preferably        85%, 90%, 95% and 98% identity after optimal alignment with        sequences SEQ ID N^(o) 13, 14 and 15, respectively,    -   A monoclonal antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially three, of        CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID N^(o)        16, 17 and 18, respectively, or sequences with at least 80%,        preferably 85%, 90%, 95% and 98% identity after optimal        alignment with sequences SEQ ID N^(o) 16, 17 and 18,        respectively, and a light chain comprising at least one,        preferentially at least two, preferentially three, of CDR-L1,        CDR-L2 and CDR-L3 of amino acid sequences SEQ ID N^(o) 19, 20        and 21, respectively, or sequences with at least 80%, preferably        85%, 90%, 95% and 98% identity after optimal alignment with        sequences SEQ ID N^(o) 19, 20 and 21, respectively,    -   A monoclonal antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially three, of        CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID N^(o)        22, 23 and 24, respectively, or sequences with at least 80%,        preferably 85%, 90%, 95% and 98% identity after optimal        alignment with sequences SEQ ID N^(o) 22, 23 and 24,        respectively, and a light chain comprising at least one,        preferentially at least two, preferentially three, of CDR-L1,        CDR-L2 and CDR-L3 of amino acid sequences SEQ ID N^(o) 25, 26        and 27, respectively, or sequences with at least 80%, preferably        85%, 90%, 95% and 98% identity after optimal alignment with        sequences SEQ ID N^(o) 25, 26 and 27, respectively,    -   A monoclonal antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially at least        three, of CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ        ID N^(o) 28, 29 and 30, respectively, or sequences with at least        80%, preferably 85%, 90%, 95% and 98% identity after optimal        alignment with sequences SEQ ID N^(o) 28, 29 and 30,        respectively, and a light chain comprising at least one,        preferentially at least two, preferentially three, of CDR-L1,        CDR-L2 and CDR-L3 of amino acid sequences SEQ ID N^(o) 31, 32        and 33, respectively, or sequences with at least 80%, preferably        85%, 90%, 95% and 98% identity after optimal alignment with        sequences SEQ ID N^(o) 31, 32 and 33, respectively    -   A monoclonal antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially three, of        CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID N^(o)        34, 35 and 36, respectively, or sequences with at least 80%,        preferably 85%, 90%, 95% and 98% identity after optimal        alignment with sequences SEQ ID N^(o) 34, 35 and 36,        respectively, and a light chain comprising at least one,        preferentially at least two, preferentially three, of CDR-L1,        CDR-L2 and CDR-L3 of amino acid sequences SEQ ID N^(o) 37, 38        and 39, respectively, or sequences with at least 80%, preferably        85%, 90%, 95% and 98% identity after optimal alignment with        sequences SEQ ID N^(o) 37, 38 and 39, respectively, and    -   A monoclonal antibody produced by the hybridoma deposited at the        CNCM, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris        CEDEX 15, France, on 27 Dec. 2016, under reference 1-5158.

As used herein, the “percentage identity” or “% identity” between twosequences of nucleic acids or amino acids refers to the percentage ofidentical nucleotides or amino acid residues between the two sequencesto be compared, obtained after optimal alignment, this percentage beingpurely statistical and the differences between the two sequences beingdistributed randomly along their length. The comparison of two nucleicacid or amino acid sequences is traditionally carried out by comparingthe sequences after having optimally aligned them, said comparison beingable to be conducted by segment or by using an “alignment window”.Optimal alignment of the sequences for comparison can be carried out, inaddition to comparison by hand, by means of methods known by a manskilled in the art.

For the amino acid sequence exhibiting at least 80%, preferably 85%,90%, 95% and 98% identity with a reference amino acid sequence,preferred examples include those containing the reference sequence,certain modifications, notably a deletion, addition or substitution ofat least one amino acid, truncation or extension. In the case ofsubstitution of one or more consecutive or non-consecutive amino acids,substitutions are preferred in which the substituted amino acids arereplaced by “equivalent” amino acids. Here, the expression “equivalentamino acids” is meant to indicate any amino acids likely to besubstituted for one of the structural amino acids without howevermodifying the biological activities of the corresponding antibodies andof those specific examples defined below.

Equivalent amino acids can be determined either on their structuralhomology with the amino acids for which they are substituted or on theresults of comparative tests of biological activity between the variousantibodies likely to be generated.

In another particular embodiment, the antibody used in the method of theinvention is a humanised antibody.

As used herein, “humanised antibody” refers to an antibody that containsCDR regions derived from an antibody of nonhuman origin, the other partsof the antibody molecule being derived from one or several humanantibodies. In addition, some of the skeleton segment residues (calledFR for framework) can be modified if necessary to preserve bindingaffinity, by using techniques well known to the person of skill in theart (Jones et al., Nature, 321:522-525, 1986). The goal of humanisationis a reduction in the immunogenicity of a xenogenic antibody, such as amurine antibody, for introduction into a human, while maintaining thefull antigen binding affinity and specificity of the antibody.

Antibodies can be humanized using a variety of techniques includingCDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101; and5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519 596; PadlanE. A., 1991, Molecular Immunology 28(4/5): 489-498; Studnicka G. M. etal., 1994, Protein Engineering 7(6): 805-814; Roguska M. A. et al.,1994, Proc. Natl. Acad. ScL U.S.A., 91:969-973), and chain shuffling(U.S. Pat. No. 5,565,332). Human antibodies can be made by a variety ofmethods known in the art including phage display methods. See also U.S.Pat. Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; andinternational patent application publication numbers WO 98/46645, WO98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO91/10741.

In a more particular embodiment, the antibody used in the method of theinvention is a humanised antibody chosen in the group consisting of:

-   -   A humanised antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially three, of        CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID N^(o)        4, 5 and 6, respectively, or sequences with at least 80%,        preferably 85%, 90%, 95% and 98% identity after optimal        alignment with sequences SEQ ID N^(o) 4, 5 and 6, respectively,        and a light chain comprising at least one, preferentially at        least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of        amino acid sequences SEQ ID N^(o) 7, 8 and 9, respectively, or        sequences with at least 80%, preferably 85%, 90%, 95% and 98%        identity after optimal alignment with sequences SEQ ID N^(o) 7,        8 and 9, respectively,    -   A humanised antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially three, of        CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID N^(o)        10, 11 and 12, respectively, or sequences with at least 80%,        preferably 85%, 90%, 95% and 98% identity after optimal        alignment with sequences SEQ ID N^(o) 10, 11 and 12,        respectively, and a light chain comprising at least one,        preferentially at least two, preferentially three, of CDR-L1,        CDR-L2 and CDR-L3 of amino acid sequences SEQ ID N^(o) 13, 14        and 15, respectively, or sequences with at least 80%, preferably        85%, 90%, 95% and 98% identity after optimal alignment with        sequences SEQ ID N^(o) 13, 14 and 15, respectively,    -   A humanised antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially three, of        CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID N^(o)        16, 17 and 18, respectively, or sequences with at least 80%,        preferably 85%, 90%, 95% and 98% identity after optimal        alignment with sequences SEQ ID N^(o) 16, 17 and 18,        respectively, and a light chain comprising at least one,        preferentially at least two, preferentially three, of CDR-L1,        CDR-L2 and CDR-L3 of amino acid sequences SEQ ID N^(o) 19, 20        and 21, respectively, or sequences with at least 80%, preferably        85%, 90%, 95% and 98% identity after optimal alignment with        sequences SEQ ID N^(o) 19, 20 and 21, respectively,    -   A humanised antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially three, of        CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID N^(o)        22, 23 and 24, respectively, or sequences with at least 80%,        preferably 85%, 90%, 95% and 98% identity after optimal        alignment with sequences SEQ ID N^(o) 22, 23 and 24,        respectively, and a light chain comprising at least one,        preferentially at least two, preferentially three, of CDR-L1,        CDR-L2 and CDR-L3 of amino acid sequences SEQ ID N^(o) 25, 26        and 27, respectively, or sequences with at least 80%, preferably        85%, 90%, 95% and 98% identity after optimal alignment with        sequences SEQ ID N^(o) 25, 26 and 27, respectively,    -   A humanised antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially three, of        CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID N^(o)        28, 29 and 30, respectively, or sequences with at least 80%,        preferably 85%, 90%, 95% and 98% identity after optimal        alignment with sequences SEQ ID N^(o) 28, 29 and 30,        respectively, and a light chain comprising at least one,        preferentially at least two, preferentially three, of CDR-L1,        CDR-L2 and CDR-L3 of amino acid sequences SEQ ID N^(o) 31, 32        and 33, respectively, or sequences with at least 80%, preferably        85%, 90%, 95% and 98% identity after optimal alignment with        sequences SEQ ID N^(o) 31, 32 and 33, respectively, and    -   A humanised antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially three, of        CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID N^(o)        34, 35 and 36, respectively, or sequences with at least 80%,        preferably 85%, 90%, 95% and 98% identity after optimal        alignment with sequences SEQ ID N^(o) 34, 35 and 36,        respectively, and a light chain comprising at least one,        preferentially at least two, preferentially three, of CDR-L1,        CDR-L2 and CDR-L3 of amino acid sequences SEQ ID N^(o) 37, 38        and 39, respectively, or sequences with at least 80%, preferably        85%, 90%, 95% and 98% identity after optimal alignment with        sequences SEQ ID N^(o) 37, 38 and 39, respectively,        wherein said antibody also comprises constant regions of the        light-chain and the heavy-chain derived from a human antibody.

In a first embodiment, a method according to the invention comprisescontacting a biological sample with at least one anti-hPG antibodybinding to an epitope of hPG, preferably one anti-hPG antibody bindingto an epitope of hPG, wherein said epitope is located within theC-terminal part of hPG. Alternatively, the method according to theinvention comprises contacting a biological sample with at least oneanti-hPG antibody binding to an epitope of hPG, preferably one anti-hPGantibody binding to an epitope of hPG, wherein said epitope is locatedwithin the N-terminal part of hPG.

In a more specific embodiment, a method according to the inventioncomprises contacting a biological sample with at least one anti-hPGantibody binding to an epitope of hPG, preferably one anti-hPG antibodybinding to an epitope of hPG, wherein said epitope includes an aminoacid sequence corresponding to an amino acid sequence of the N-terminalpart of progastrin chosen among an amino acid sequence corresponding toamino acids 10 to 14 of hPG, amino acids 9 to 14 of hPG, amino acids 4to 10 of hPG, amino acids 2 to 10 of hPG and amino acids 2 to 14 of hPG,wherein the amino acid sequence of hPG is SEQ ID N^(o) 1.

In a more specific embodiment, a method according to the inventioncomprises contacting a biological sample with at least one anti-hPGantibody binding to an epitope of hPG, preferably one anti-hPG antibodybinding to an epitope of hPG, wherein said epitope includes an aminoacid sequence corresponding to an amino acid sequence of the C-terminalpart of progastrin, chosen among an amino acid sequence corresponding toamino acids 71 to 74 of hPG, amino acids 69 to 73 of hPG, amino acids 71to 80 of hPG (SEQ ID N^(o) 40), amino acids 76 to 80 of hPG, and aminoacids 67 to 74 of hPG, wherein the amino acid sequence of hPG is SEQ IDN^(o) 1.

In a particular embodiment of the method of the invention, said methodcomprises a step of contacting a biological sample from a subject with afirst agent which binds to a first part of progastrin and with a secondagent which binds to a second part of progastrin. In a more particularembodiment, wherein said progastrin-binding molecule is an antibody, abiological sample from a subject is contacted with an antibody whichbinds to a first epitope of progastrin and with a second antibody whichbinds to a second epitope of progastrin.

According to a preferred embodiment, said first antibody is bound to aninsoluble or partly soluble carrier. Binding of progastrin by said firstantibody results in capture of progastrin from said biological sample.Preferably, said first antibody is an antibody binding to an epitope ofhPG, wherein said epitope includes an amino acid sequence correspondingto an amino acid sequence of the C-terminal part of progastrin, asdescribed above. More preferably, said first antibody is monoclonalantibody Mab14, produced by hybridoma 2H9F4B7, described in WO2011/083088. Hybridoma 2H9F4B7 was deposited under the Budapest Treatyat the CNCM, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 ParisCEDEX 15, France, on 27 Dec. 2016, under reference I-5158.

According to another preferred embodiment, said second antibody islabelled with a detectable moiety, as described below. Binding ofprogastrin by second antibody enables the detection of the progastrinmolecules which were present in the biological sample. Further, bindingof progastrin by second antibody enables the quantification of theprogastrin molecules which were present in the biological sample.Preferably, said second antibody is an antibody binding to an epitope ofhPG, wherein said epitope includes an amino acid sequence correspondingto an amino acid sequence of the N-terminal part of progastrin, asdescribed above. More preferably, said N-terminal antibody is apolyclonal antibody, as described above. Alternatively, it is alsopossible to use a monoclonal antibody biding an epitope within theN-terminus of progastrin, such as e.g. the N-terminus monoclonalantibodies described above, notably a monoclonal antibody comprising aheavy chain comprising CDR-H1, CDR-H2 and CDR-H3 of amino acid sequencesSEQ ID N^(o) 16, 17 and 18, respectively, and a light chain comprisingCDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID N^(o) 19, 20and 21.

In a particularly preferred embodiment, the first antibody is bound toan insoluble or partly soluble carrier and the second antibody islabelled with a detectable moiety.

In a particular embodiment, the method of the present inventioncomprises the determination of the concentration of progastrin in abiological sample from a human subject who has never been previouslydiagnosed with cancer.

In another particular embodiment, the method of the present inventioncomprises the determination of the concentration of progastrin in abiological sample from a human subject who has never been previouslydiagnosed with cancer, wherein said biological sample is selected fromblood, serum and plasma.

In a more particular embodiment, the method of the present inventioncomprises contacting a plasma sample from said subject with at least oneanti-hPG antibody, notably with one anti-hPG antibody, and determiningthe concentration of progastrin in said sample, wherein a concentrationof progastrin superior to 10 pM in said plasma is indicative of a riskof developing cancer in said subject. In other words, a concentration ofprogastrin superior to 10 pM in said plasma is indicative of a badprognosis in said subject.

Still more preferably, the method of the present invention comprisescontacting a plasma sample from said subject with at least one anti-hPGantibody, notably with one anti-hPG antibody, and determining theconcentration of progastrin in said sample, wherein a concentration ofprogastrin superior to 10 pM, preferably to 20 pM, more preferably to 30pM, still more preferably to 40 pM, even more preferably to 50 pM insaid plasma sample is indicative of a risk of developing cancer in saidsubject.

In another aspect, the present invention relates to a method of treatinga cancer in a patient who had never been diagnosed for cancer, saidmethod comprising the steps of:

-   -   a) evaluating the risk of said patient to develop a cancer by        any of the methods described above; and    -   b) treating said cancer if there is risk according to a).

The method of the invention is particularly advantageous, because itallows cancers to be identified at a very early stage. It is known inthe art that the earlier the identification of the cancer, the higherthe chances of remission. In addition, the patient can be treated withanti-cancer drugs which are not too aggressive, thus lessening thechances of side-effects whilst maintaining therapeutic efficiency.

In a particular embodiment, a method according to the inventioncomprises comparing the concentration of progastrin in a biologicalsample obtained from a patient with a predetermined value ofconcentration of progastrin in the sample, in a more particularembodiment, said predetermined value is chosen among: a mean, oraverage, of sample values based on the mean, or average, determinationof the value in a population free of cancer, a progastrin concentrationvalue obtained when the patient was known to be free of cancer.

In yet another aspect, the invention also provides a composition for usein the methods described above. According to this aspect of theinvention, the composition is for evaluating the risk of the occurrenceof cancer in a subject who has never been diagnosed previously withcancer, wherein said composition comprises at least oneprogastrin-binding antibody, or an antigen-binding fragment thereof.

In a first embodiment, a composition according to the inventioncomprises an antibody recognizing an epitope including an amino acidsequence corresponding to an amino acid sequence of progastrin.

In a more specific embodiment, a composition according to the inventioncomprises an antibody recognizing an epitope of progastrin wherein saidepitope includes an amino acid sequence corresponding to an amino acidsequence of the N-terminal part of progastrin, wherein said amino acidsequence may include residues 10 to 14 of hPG, residues 9 to 14 of hPG,residues 4 to 10 of hPG, residues 2 to 10 of hPG or residues 2 to 14 ofhPG, wherein the amino acid sequence of hPG is SEQ ID N^(o) 1.

In a more specific embodiment, a composition according to the inventioncomprises an antibody recognizing an epitope of progastrin wherein saidepitope includes an amino acid sequence corresponding to an amino acidsequence of the C-terminal part of progastrin, wherein said amino acidsequence may include residues 71 to 74 of hPG, residues 69 to 73 of hPG,residues 71 to 80 of hPG (SEQ ID N^(o) 40), residues 76 to 80 of hPG, orresidues 67 to 74 of hPG, wherein the amino acid sequence of hPG is SEQID N^(o) 1.

In still another aspect, the present invention provides a kit useful forthe methods described above, said kit comprising any of the antibodiesof the invention. Packaged materials comprising a combination ofreagents in predetermined amounts with instructions for performing themethods described above, e.g. kits, are also within the scope of theinvention. Preferably, said kit comprises at least one antibody of theinvention, more preferably two.

For example, in a first embodiment, said kit comprises a first antibodybound to an insoluble or partly soluble carrier. Preferably, said firstantibody is an antibody binding to an epitope of hPG, wherein saidepitope includes an amino acid sequence corresponding to an amino acidsequence within the C-terminal part of progastrin, as described above.More preferably, said first antibody is monoclonal antibody Mab14,produced by hybridoma 2H9F4B7, described in WO 2011/083088. Hybridoma2H9F4B7 was deposited under the Budapest Treaty at the CNCM, InstitutPasteur, 25-28 rue du Docteur Roux, 75724 Paris CEDEX 15, France, on 27Dec. 2016, under reference 1-5158. In another embodiment, polyclonal ormonoclonal antibodies, or antigen-binding fragment or derivativethereof, as detailed herein are provided labeled with a detectablemoiety, such that they may be packaged and used, for example, in kits,to identify cells having the aforementioned antigen, either beforesecretion or bound to the receptor for progastrin. Non-limiting examplesof such labels include fluorophores such as fluorescein isothiocyanate;chromophores, radionuclides, biotin or enzymes. Such labeled antibodiesor binding fragments may be used for the histological localization ofthe antigen, ELISA, cell sorting, as well as other immunologicaltechniques for detecting or quantifying progastrin, and cells bearingthis antigen, for example. Preferably, said labeled antibody is anantibody binding to an epitope of hPG, wherein said epitope includes anamino acid sequence corresponding to an amino acid sequence within theN-terminal part of progastrin, as described above. More preferably, saidN-terminal antibody is a polyclonal antibody, as described above.Alternatively, it is also possible to use a monoclonal antibody bidingan epitope within the N-terminus of progastrin, such as e.g. theN-terminus monoclonal antibodies described above, notably a monoclonalantibody comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3of amino acid sequences SEQ ID N^(o) 16, 17 and 18, respectively, and alight chain comprising CDR-L1, CDR-L2 and CDR-L3 of amino acid sequencesSEQ ID N^(o) 19, 20 and 21.

Thus in a most preferred embodiment, the kit of the invention comprises:

-   -   a first anti-progastrin antibody, wherein said antibody is        wherein said first anti-progastrin antibody is a monoclonal        antibody produced by the hybridoma deposited at the CNCM,        Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris CEDEX        15, France, on 27 Dec. 2016, under reference 1-5158; and    -   a second anti-progastrin antibody, wherein said second        anti-progastrin antibody is a polyclonal antibody binding an        epitope within the N-terminus of progastrin or a monoclonal        antibody comprising a heavy chain comprising the following three        CDRs, CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID        N^(o) 16, 17 and 18, respectively, and a light chain comprising        the following three CDRs, CDR-L1, CDR-L2 and CDR-L3 of amino        acid sequences SEQ ID N^(o) 19, 20 and 21, respectively.

The invention includes kits wherein the antibody, or antigen-bindingfragment or derivative thereof, is labeled.

The reagents may be provided as dry powders, usually lyophilized,including excipients which on dissolution will provide a reagentsolution having the appropriate concentration.

The kit contains the antibodies for detection and quantification ofprogastrin in vitro, e.g. in an ELISA or a Western blot. The antibody ofthe present invention can be provided in a kit for detection andquantification of progastrin in vitro, e.g. in an ELISA or a Westernblot. Where the antibody is labeled with an enzyme, the kit will includesubstrates and cofactors required by the enzyme (e.g., a substrateprecursor which provides the detectable chromophore or fluorophore). Inaddition, other additives may be included such as stabilizers, buffers(e.g., a block buffer or lysis buffer) and the like. Such a kit maycomprise a receptacle being compartmentalized to receive one or morecontainers such as vials, tubes and the like, such containers holdingseparate elements of the invention. For example, one container maycontain a first antibody bound to an insoluble or partly solublecarrier. A second container may contain soluble, detectably-labeledsecond antibody, in lyophilized form or in solution. The receptacle mayalso contain a third container holding a detectably labeled thirdantibody in lyophilized form or in solution. A kit of this nature can beused in the sandwich assay of the invention. The label or package insertmay provide a description of the composition as well as instructions forthe intended in vitro or diagnostic use.

Kits are also provided for use as a positive control for purification orimmunoprecipitation of progastrin from cells. For isolation andpurification of progastrin, the kit can contain the antibody describedherein, or an antigen-binding fragment or derivative thereof, coupled tobeads (e.g., sepharose beads). Kits can be provided which contain theantibodies for detection and quantification of progastrin in vitro or exvivo, e.g. in an ELISA or a Western blot. The kit comprises a containerand a label or package insert on or associated with the container. Thecontainer holds a composition comprising at least one antibody, orbinding fragment or derivative thereof, of the invention. Additionalcontainers may be included that contain, e.g., diluents and buffers,control antibodies. The label or package insert may provide adescription of the composition as well as instructions for the intendedin vitro or diagnostic use.

The invention also relates to a product/computer program containing aset of instructions characteristic of the implementation of theinventive method.

The invention also relates to a processing system including acomputation unit and an input interface, characterized in that saidsystem includes means for implementing the method for determining therisk of developing cancer as disclosed herein.

In reference to FIG. 13, a device (1) according to a particularembodiment of the present invention includes a computation unit (10)capable of following computer instructions and processing data. One suchcomputation unit preferentially includes a microprocessor (110), whichcan be of any type known in the state of the art. The computation unit(10) also has a storage unit (100) that is capable of receiving acomputer program including a set of instructions characteristic of theimplementation of the method, and is capable of storing data.

The device (1) also includes an input interface (12) connected to thecomputation unit (10) enabling an operator (O) of the device (1) toenter data to be treated. One such input interface (12) includes anyelement enabling the entry of such data destined for the computationunit (10) such as a keyboard element optionally associated with apointing device element.

Preferentially, the computation unit further includes an outputinterface (14) such as a screen that on the one hand enables the user toverify the integrity of the data entered but on the other hand enablesthe computation unit (10) to be able to interact with the operator (O).

The device (1) can be integrated in a single system such as a computer,a smartphone or any other system known in the state of the art enablingimplementation of the inventive method. The operator (O) can be of anyskill level and thus may or may not have medical qualifications.

It is notably envisaged according to a particular embodiment of thepresent invention that the data entered by the operator (O) are sent viaa network (the Internet, for example) preferentially in a secure mannerto a remote server comprising a computation unit capable of implementingthe inventive method and thus of treating the data received by theserver. Optionally, after said processing, the server returns the resultof the analysis to the user via the same network or another. Optionally,the server records the data and/or the result of the analysis on a meansof recording.

Obviously, means of guaranteeing the anonymity of thephysiological/clinical characteristics of the donor and the recipientcan be envisaged.

Thus, one such device (1) enables implementation of the inventivemethod, i.e., it enables implementation of the following steps:

-   -   entering physiological/clinical characteristics using the input        interface (12) into a computation unit (10) (step 22), said        characteristics including the level of progastrin in the sample        (10) (step 23),    -   optionally normalizing said progastrin level via data processing        by the computation unit (10) (step 24), and    -   analyzing said risk score so as to determine risk of development        of cancer (step 25).

Thus, the method of the invention can be implemented not only byclinical or hospital personnel but also by all persons involved inclinical research (pharmaceutical industry, scientists, doctors, etc.)or even by the general public.

The examples that follow are merely exemplary of the scope of thisinvention and content of this disclosure. One skilled in the art candevise and construct numerous modifications to the examples listed belowwithout departing from the scope of this invention.

FIGURE LEGENDS

FIG. 1: Receiver operating characteristic (ROC) curve for colorectalcancer (upper panel) with the area under the ROC curve and thestatistical analysis (lower panel).

FIG. 2: median plasmatic concentration of progastrin in colorectalcancer patients (n=148), and in control patients (n=103) using acombination of an N-terminus polyclonal antibody and a C-terminuspolyclonal antibody—Mann Whitney test two-tailed, *** p<0.0001.

FIG. 3: Receiver operating characteristic (ROC) curve for hepatocellularcarcinoma (upper panel) with the area under the ROC curve and thestatistical analysis (lower panel).

FIG. 4: median plasmatic concentration of progastrin in hepatocellularcarcinoma patients (n=47), and in control patients (n=103) using acombination of an N-terminus polyclonal antibody and a C-terminuspolyclonal antibody—Mann Whitney test two-tailed, *** p<0.0001

FIG. 5: Receiver operating characteristic (ROC) curve for oesophagiccancer (upper panel) with the area under the ROC curve and thestatistical analysis (lower panel).

FIG. 6: median plasmatic concentration of progastrin in oesophagiccancer patients (n=12), and in control patients (n=103) using acombination of an N-terminus polyclonal antibody and a C-terminuspolyclonal antibody—Mann Whitney test two-tailed, *** p<0.0001

FIG. 7: Receiver operating characteristic (ROC) curve for gastric cancer(upper panel) with the area under the ROC curve and the statisticalanalysis (lower panel).

FIG. 8: median plasmatic concentration of progastrin in gastric cancerpatients (n=15), and in control patients (n=103) using a combination ofan N-terminus polyclonal antibody and a C-terminus polyclonalantibody—Mann Whitney test two-tailed, p<0.001

FIG. 9: Receiver operating characteristic (ROC) curve for pancreaticcancer (upper panel) with the area under the ROC curve and thestatistical analysis (lower panel).

FIG. 10: median plasmatic concentration of progastrin in pancreaticcancer patients (n=44), and in control patients (n=103) using acombination of an N-terminus polyclonal antibody and a C-terminuspolyclonal antibody—Mann Whitney test two-tailed, *** p<0.0001

FIG. 11: Receiver operating characteristic (ROC) curve for ovariancancer (upper panel) with the area under the ROC curve and thestatistical analysis (lower panel).

FIG. 12: median plasmatic concentration of progastrin in ovarian cancerpatients (n=8), and in control patients (n=103) using a combination ofan N-terminus polyclonal antibody and a C-terminus polyclonal antibody.

FIG. 13: Schematic representation of a processing system according to aparticular embodiment of the present invention

FIG. 14: Functional graph representing a method according to aparticular embodiment of the present invention.

FIG. 15: Median plasmatic concentration of progastrin in various cancertype patients (n=231), and in control patients (n=322) using acombination of a polyclonal antibody and a monoclonal antibody.

FIG. 16: Median plasmatic concentration of progastrin in various cancertype patients (n=10), using a combination of a polyclonal antibody andmonoclonal antibody (mAb-pAb) or a combination of monoclonal antibodies(mAb-mAb)—Mann Whitney test two-tailed, NS p>0.05.

EXAMPLES Example 1: Detection of Plasmatic Progastrin ConcentrationUsing Polyclonal Antibodies

Plasma progastrin levels were quantified by ELISA through the use of twospecific anti-progastrin antibodies: capture antibodies are coated onthe wells of the plate, whereas revelation antibodies are used to detectprogastrin and mediates revelation of the signal.

In the present example, quantification is based on the ELISA methodwhich allows, through the use of a substrate whose reaction emits light,to assign a value proportional to the luminescence amount of antibodiesbound to the antigen retained by capture antibodies.

Material

Reagents and apparatus are listed in Table 7:

TABLE 7 Designation Provider Référence Plates MaxiSORP white Nunc, 96wells Dutscher #055221 Sodium Carbonate/Bicarbonate Sigma #21851 DPBS 1XLonza #P04-36500 Tween-20 Biosolve #20452335 BSA Euromedex #04-100-810-C Streptavidin-HRP Pierce #21130 (Thermo) SuperSignal ELISA FemtoMaximum Pierce #37074 Sensitivity Substrate (Thermo) Anti-ProGastrinPolyclonal Antibody Eurogentec /

Polyclonal antibodies were obtained by immunizing a rabbit withN-terminal progastrin (SEQ ID N^(o) 2) or with C-terminal progastrincorresponding to amino acids 71 to 80 of hPG and having the sequenceFGRRSAEDEN (SEQ ID N^(o) 40), according to standard protocols.

The binding characteristics of polyclonal antibodies against progastrinused in this assay are the following: absence of binding to G34-Gly,G34, G17-Gly, G17, binding to full length progastrin.

96 wells plates are coated by preparing a solution of carbonate—sodiumbicarbonate, 50 mM pH 9.6 by dissolving the contents of one capsule in100 ml of MilliQ water. A solution of capture antibody (3 μg/ml),corresponding to polyclonal antibodies obtained by using the C-terminalof progastrin FGRRSAEDEN (SEQ ID N^(o) 40) is prepared in carbonatebuffer. 100 microliters of antibodies solution is added to each well andincubated at 4° C. for 16 hours (1 night). Plates are then blocked byeliminating the antibodies solution and wash 3 times with 300 μl1×PBS/0.1% Tween-20, then adding 200 μl of blocking buffer (1×PBS/0.1%Tween-20/0.1% BSA) per well, and incubated 2 hours at 22° C. Blockingbuffer is then eliminated, wells are washed 3 times with 300 μl1×PBS/0.1% Tween-20.

Plasma dilution is performed as follows: The plasma is used pure,diluted 1/2, 1/5 and 1/10. Dilutions are prepared from pure plasma in1×PBS/0.1% Tween 20/0.1% BSA.

For the control test, ELISA in the presence of a known concentration ofprogastrin, progastrin dilution is prepared as follows: stockrecombinant PG (Full length human progastrin produced in E. coli andaffinity purified with Glutathione agarose/Tag removal (Tev)/IMACCounter purification/dialysis, from Institut Pasteur, Paris, France) isprepared at a concentration of 0.45 mg/ml (45 microM), in triplicate.Ranges of progastrin concentrations were prepared as follows:

-   -   Solution A: Pre-dilution 1/10, 2 μl of stock+18 μl of the buffer    -   Solution B: Pre-dilution 1/100, 10 μl of A+90 μl of the buffer    -   Solution C: Pre-dilution 1/1000, 10 μl of B+90 μl of the buffer    -   Solution D: 500 pM, 5.55 μl of C+494.5 μl of the diluent    -   Solution E: 250 pM, 250 μl of D+250 μl of the diluent    -   Solution F: 100 pM, 200 μl of E+300 μl of the diluent    -   Solution G: 50 pM, 250 μl of F+250 μl of the diluent    -   Solution H: 25 pM, 200 μl of G+200 μl of the diluent    -   Solution I: 10 pM, 100 μl of H+150 μl of the diluent

The range of recombinant PG is linear and can therefore be more or lessextensive according to the antibody used.

For the preparation of test samples, approximately 500 μl of each sampleare set aside and stored until analysis (and confirmation if necessary)of the results. 100 μl of each point of the range and/or plasmas areassayed pure, diluted to 1/2, 1/5 and 1/10, and incubated for 2 hours at22° C. on the plates.

For the revelation of the test, the plates are washed 3 times with 300μl 1×PBS/0.1% Tween-20. A solution of the polyclonal rabbitanti-progastrin antibody, wherein said antibodies have been obtained byusing the N-terminal part of progastrin as an immunogen, coupled tobiotin to 0.5 μg/ml, is prepared by dilution in 1×PBS/0.1% Tween-20/0.1%BSA. 100 μl of this solution is added to each well. Incubation takesplace for 1 hour at 22° C. The revelation with streptavidin-HRP isperformed by removing detection antibody and wash 3 times with 300 μl1×PBS/0.1% Tween-20, then preparing a solution of Streptavidin-HRP at 20ng/ml diluted in 1×PBS/0.1% Tween-20/0.1% BSA, wherein 100 Add 100 μl ofthis solution is added to each well, before incubation for 1 hour at 22°C.

The detection consists of eliminating streptavidin-HRP and wash 3 timeswith 300 μl 1×PBS/0.1% Tween-20, then adding 100 μl of chemiluminescentsubstrate solution per well. The substrate solution is prepared bymixing equal volumes of the two solutions SuperSignal ELISA Femto kit,20 ml+20 ml, 30 minutes before use and stored at room temperature in thedark. Luminescence is read after 5 minutes incubation at roomtemperature in the dark.

For each condition, the test is performed in triplicate and the resultsof the ranges will be presented as a graph showing the change inluminescence depending on the progastrin concentration. For each plasmadilution, the concentration of progastrin is determined using theequation of the linear regression line of the corresponding range (range1/10th for a sample diluted to 1/10th).

Methods and Results

Progastrin levels were determined in plasma samples from subjects whowere known to have developed cancer later. Progastrin was captured withpolyclonal antibodies specific for the C-terminus. Detection wasperformed with labelled polyclonal antibodies specific for theN-terminus.

Importantly, at the time of sample collection, these subjects had neverbeen diagnosed with cancer and did not show any symptom relating tocancer. The control was constituted by plasma samples from the generalpopulation.

The results are shown in FIGS. 1-12. The median plasmatic concentrationof progastrin was 17 pM in patients who developed colorectal cancerafterwards (n=148), 100 pM in patients who developed hepatocellularcarcinoma (n=47), 42.3 pM in patients who developed oesophagic cancer(n=12), 17.90 pM in patients who developed gastric cancer (n=15), 16.6pM in patients who developed pancreatic cancer (n=44), and 8.45 inpatients who developed ovarian cancer (n=8). By comparison, the medianplasmatic concentration of progastrin is 0 pM in control subjects(n=103).

These data demonstrate that patients who will develop cancer havedetectable levels of progastrin in their plasma whereas healthy controlindividuals have none. Progastrin can be detected even before any cancercan be diagnosed, making progastrin useful biomarker for the onset ofcancer. ROC analysis confirmed the predictive nature of progastrin foreach of the above-listed cancers.

These data demonstrate that patients with a risk of developing cancerhave higher concentration of progastrin in their plasma compared tohealthy control individuals.

Example 2: Detection of Plasmatic Progastrin Concentration Using aCombination of Polyclonal Antibodies and Monoclonal Antibodies

In the present example, plasma progastrin levels were quantified byELISA through the use of antibody specific for human progastrin (hPG)pre-coated on a 96-well plate. Standards and samples are added to thewells, and any hPG present binds to the immobilized capture antibody.The wells are washed and an anti-hPG detection antibody horseradishperoxidase (HRP) conjugate is added, producing anantibody-antigen-antibody “sandwich.” After a second wash, TMB substratesolution is added, which produces a blue color in direct proportion tothe amount of hPG present in the initial sample. The Stop Solutionchanges the color from blue to yellow, and the wells are read at 450 nmwith a microplate reader.

Polyclonal antibodies were obtained by immunizing a rabbit withN-terminal progastrin (SEQ ID N^(o) 2) or with C-terminal progastrincorresponding to amino acids 71 to 80 of hPG and having the sequenceFGRRSAEDEN (SEQ ID N^(o) 40), according to standard protocols.

Monoclonal antibodies were obtained by using hybridomas producingantibodies against N-terminal progastrin (SEQ ID N^(o) 2) or againstC-terminal progastrin corresponding to amino acids 71 to 80 of hPG andhaving the sequence FGRRSAEDEN (SEQ ID N^(o) 40), according to standardprotocols.

The binding characteristics of polyclonal and monoclonal antibodiesagainst progastrin used in this assay are the following: absence ofbinding to G34-Gly, G34, G17-Gly, G17, binding to full lengthprogastrin.

For the control test, ELISA in the presence of a known concentration ofprogastrin, progastrin dilution is prepared as follows: stockrecombinant PG (Full length human progastrin produced in E. coli andaffinity purified with Glutathione agarose/Tag removal (Tev)/IMACCounter purification/dialysis, from Institut Pasteur, Paris, France) isprepared at a concentration of 0.45 mg/ml (45 microM), in triplicate.Ranges of progastrin concentrations were prepared as follows:

-   -   Solution A: Pre-dilution 1/10, 2 μl of stock+18 μl of the buffer    -   Solution B: Pre-dilution 1/100, 10 μl of A+90 μl of the buffer    -   Solution C: Pre-dilution 1/1000, 10 μl of B+90 μl of the buffer    -   Solution D: 500 pM, 5.55 μl of C+494.5 μl of the diluent    -   Solution E: 250 pM, 250 μl of D+250 μl of the diluent    -   Solution F: 100 pM, 200 μl of E+300 μl of the diluent    -   Solution G: 50 pM, 250 μl of F+250 μl of the diluent    -   Solution H: 25 pM, 200 μl of G+200 μl of the diluent    -   Solution I: 10 pM, 100 μl of H+150 μl of the diluent

The range of recombinant PG is linear and can therefore be more or lessextensive according to the antibody used.

Methods and Results

Progastrin levels were determined in plasma samples from subjects whowere known to have developed cancer later. Progastrin was captured withthe C-terminus monoclonal antibody mAb 14 produced by hybridoma 2H9F4B7described in WO 2011/083088 (Hybridoma 2H9F4B7 was deposited under theBudapest Treaty at the CNCM, Institut Pasteur, 25-28 rue du DocteurRoux, 75724 Paris CEDEX 15, France, on 27 Dec. 2016, under reference1-5158.). Detection was performed with labelled polyclonal antibodiesspecific for the N-terminus.

Importantly, at the time of sample collection, these subjects had neverbeen diagnosed with cancer and did not show any symptom relating tocancer. The control was constituted by plasma samples from the generalpopulation.

The results are shown in FIG. 15. The median plasmatic concentration ofprogastrin was ranked between 2.750 and 21.5 pM in patients depending ofthe type of cancer (n=231). By comparison, the median plasmaticconcentration of progastrin is 0 pM in control subjects (n=322).

These data demonstrate that patients who will develop cancer havedetectable levels of progastrin in their plasma whereas healthy controlindividuals have none. Progastrin can be detected even before any cancercan be diagnosed, making progastrin useful biomarker for the onset ofcancer.

These data demonstrate that patients with a risk of developing cancerhave higher concentration of progastrin in their plasma compared tohealthy control individuals.

Example 3: Detection of Plasmatic Progastrin Concentration Using aCombination of Monoclonal Antibodies

In the present example, plasma progastrin levels were quantified byELISA through the use of antibody specific for human progastrin (hPG)pre-coated on a 96-well plate. Standards and samples are added to thewells, and any hPG present binds to the immobilized capture antibody.The wells are washed and an anti-hPG detection antibody horseradishperoxidase (HRP) conjugate is added, producing anantibody-antigen-antibody “sandwich.” After a second wash, TMB substratesolution is added, which produces a blue color in direct proportion tothe amount of hPG present in the initial sample. The Stop Solutionchanges the color from blue to yellow, and the wells are read at 450 nmwith a microplate reader.

Polyclonal antibodies were obtained by immunizing a rabbit withN-terminal progastrin (SEQ ID N^(o) 2) or with C-terminal progastrincorresponding to amino acids 71 to 80 of hPG and having the sequenceFGRRSAEDEN (SEQ ID N^(o) 40), according to standard protocols.

Monoclonal antibodies were obtained by using hybridomas producingantibodies against N-terminal progastrin (SEQ ID N^(o) 2) or againstC-terminal progastrin corresponding to amino acids 71 to 80 of hPG andhaving the sequence FGRRSAEDEN (SEQ ID N^(o) 40), according to standardprotocols.

The binding characteristics of polyclonal and monoclonal antibodiesagainst progastrin used in this assay are the following: absence ofbinding to G34-Gly, G34, G17-Gly, G17, binding to full lengthprogastrin.

For the control test, ELISA in the presence of a known concentration ofprogastrin, progastrin dilution is prepared as follows: stockrecombinant PG (Full length human progastrin produced in E. coli andaffinity purified with Glutathione agarose/Tag removal (Tev)/IMACCounter purification/dialysis, from Institut Pasteur, Paris, France) isprepared at a concentration of 0.45 mg/ml (45 microM), in triplicate.Ranges of progastrin concentrations were prepared as follows:

-   -   Solution A: Pre-dilution 1/10, 2 μl of stock+18 μl of the buffer    -   Solution B: Pre-dilution 1/100, 10 μl of A+90 μl of the buffer    -   Solution C: Pre-dilution 1/1000, 10 μl of B+90 μl of the buffer    -   Solution D: 500 pM, 5.55 μl of C+494.5 μl of the diluent    -   Solution E: 250 pM, 250 μl of D+250 μl of the diluent    -   Solution F: 100 pM, 200 μl of E+300 μl of the diluent    -   Solution G: 50 pM, 250 μl of F+250 μl of the diluent    -   Solution H: 25 pM, 200 μl of G+200 μl of the diluent    -   Solution I: 10 pM, 100 μl of H+150 μl of the diluent

The range of recombinant PG is linear and can therefore be more or lessextensive according to the antibody used.

Methods and Results

Progastrin levels were determined in plasma samples from subjects whowere known to have developed cancer later. Progastrin was captured withthe C-terminus monoclonal antibody mAb 14 produced by hybridoma 2H9F4B7described in WO 2011/083088 (Hybridoma 2H9F4B7 was deposited under theBudapest Treaty at the CNCM, Institut Pasteur, 25-28 rue du DocteurRoux, 75724 Paris CEDEX 15, France, on 27 Dec. 2016, under reference1-5158.). Detection was performed with labelled monoclonal antibody mAb16 described in WO 2011/083088 which is specific for the N-terminus.

Importantly, at the time of sample collection, these subjects had neverbeen diagnosed with cancer and did not show any symptom relating tocancer. The control was constituted by plasma samples from the generalpopulation.

The results are shown in FIG. 16. The median plasmatic concentration ofprogastrin using mAb-pAb et mAb-mAb were similar in median (8.2 vs 6.6pM respectively) and mean (18.99 vs 16.79 pM) respectively (n=10).

These data demonstrate that mAb-pAb and mAb-mAb ELISA sandwich testsdetect both progastrin in the plasma of patients. Importantly, nosignificant difference between both tests could be identified. Inparticular, the sensitivity of the mAb-pAb and mAb-mAb sandwiches werehighly similar. Therefore mAb-mAb ELISA sandwich can be used reliably todetect progastrin in the plasma of patient even before any cancer can bediagnosed, making progastrin useful biomarker for the onset of cancer.

These data demonstrate that patients with a risk of developing cancerhave higher concentration of progastrin in their plasma compared tohealthy control individuals.

1. A method for evaluating the risk of occurrence of a cancer in asubject who has not been previously diagnosed with cancer, said methodcomprising the steps of: a) determining the level of progastrin in asample of said subject; b) determining the risk that said subject willdevelop a cancer based on the level of step a).
 2. The method of claim1, wherein the determination of step a) includes contacting said samplewith at least one progastrin-binding molecule and measuring the bindingof said progastrin-binding molecule to progastrin.
 3. The method ofclaim 2, wherein said agent binding to progastrin is an anti-progastrinantibody, or an antigen-binding fragment thereof.
 4. The method of claim3, wherein said antibody is a monoclonal antibody or a polyclonalantibody.
 5. The method of any one of claim 3 or 4, wherein saidantibody is selected among N-terminal anti-progastrin antibodies andC-terminal anti-progastrin antibodies.
 6. The method of claims 3 to 5,wherein said antibody is a monoclonal antibody selected in the groupconsisting of: A monoclonal antibody comprising a heavy chain comprisingat least one, preferentially at least two, preferentially three, ofCDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID N^(o) 4, 5 and6, respectively, and a light chain comprising at least one,preferentially at least two, preferentially three, of CDR-L1, CDR-L2 andCDR-L3 of amino acid sequences SEQ ID N^(o) 7, 8 and 9, respectively, Amonoclonal antibody comprising a heavy chain comprising at least one,preferentially at least two, preferentially three, of CDR-H1, CDR-H2 andCDR-H3 of amino acid sequences SEQ ID N^(o) 10, 11 and 12, respectively,and a light chain comprising at least one, preferentially at least two,preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of amino acidsequences SEQ ID N^(o) 13, 14 and 15, respectively, A monoclonalantibody comprising a heavy chain comprising at least one,preferentially at least two, preferentially three, of CDR-H1, CDR-H2 andCDR-H3 of amino acid sequences SEQ ID N^(o) 16, 17 and 18, respectively,and a light chain comprising at least one, preferentially at least two,preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of amino acidsequences SEQ ID N^(o) 19, 20 and 21, respectively, A monoclonalantibody comprising a heavy chain comprising at least one,preferentially at least two, preferentially three, of CDR-H1, CDR-H2 andCDR-H3 of amino acid sequences SEQ ID N^(o) 22, 23 and 24, respectively,and a light chain comprising at least one, preferentially at least two,preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of amino acidsequences SEQ ID N^(o) 25, 26 and 27, respectively, A monoclonalantibody comprising a heavy chain comprising at least one,preferentially at least two, preferentially three, of CDR-H1, CDR-H2 andCDR-H3 of amino acid sequences SEQ ID N^(o) 28, 29 and 30, respectively,and a light chain comprising at least one, preferentially at least two,preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of amino acidsequences SEQ ID N^(o) 31, 32 and 33, respectively A monoclonal antibodycomprising a heavy chain comprising at least one, preferentially atleast two, preferentially three, of CDR-H1, CDR-H2 and CDR-H3 of aminoacid sequences SEQ ID N^(o) 34, 35 and 36, respectively, and a lightchain comprising at least one, preferentially at least two,preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of amino acidsequences SEQ ID N^(o) 37, 38 and 39, respectively, and A monoclonalantibody produced by the hybridoma deposited at the CNCM, InstitutPasteur, 25-28 rue du Docteur Roux, 75724 Paris CEDEX 15, France, on 27Dec. 2016, under reference 1-5158.
 7. The method of any one of claims 1to 6, wherein the determination of step a) includes: (i) contacting saidsample with a first progastrin-binding molecule which binds to a firstpart of progastrin, and (ii) contacting said sample with a secondprogastrin-binding molecule which binds to a second part of progastrin.8. The method of claim 7, wherein the first progastrin-binding moleculebinds an epitope within the C-terminus of progastrin.
 9. The method ofany one of claim 7 or 8, wherein said progastrin-binding molecule is amonoclonal antibody produced by the hybridoma deposited at the CNCM,Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris CEDEX 15,France, on 27 Dec. 2016, under reference 1-5158.
 10. The method of anyone of claims 7 to 9, wherein the second progastrin-binding moleculebinds an epitope within the N-terminus of progastrin.
 11. The method ofany one of claims 7 to 10, wherein said second progastrin-bindingmolecule is a polyclonal antibody binding an epitope within theN-terminus of progastrin or a monoclonal antibody comprising a heavychain comprising the following three CDRs, CDR-H1, CDR-H2 and CDR-H3 ofamino acid sequences SEQ ID N^(o) 16, 17 and 18, respectively, and alight chain comprising the following three CDRs, CDR-L1, CDR-L2 andCDR-L3 of amino acid sequences SEQ ID N^(o) 19, 20 and 21, respectively.12. The method of any one of claims 1 to 11, wherein the level ofprogastrin is determined in step a) with an ELISA.
 13. The methodaccording to any one of claims 1 to 12, wherein biological sample ischosen among: blood, serum and plasma.
 14. A kit comprising at least oneanti-progastrin antibody as defined in any one of claims 2 to 6 forevaluating the risks of developing cancer in a subject who has not beenpreviously diagnosed with cancer.
 15. The kit of claim 14, comprising: afirst anti-progastrin antibody, wherein said antibody is wherein saidfirst anti-progastrin antibody is a monoclonal antibody produced by thehybridoma deposited at the CNCM, Institut Pasteur, 25-28 rue du DocteurRoux, 75724 Paris CEDEX 15, France, on 27 Dec. 2016, under reference1-5158; and a second anti-progastrin antibody, wherein said secondanti-progastrin antibody is a polyclonal antibody binding the N-terminusof progastrin or a monoclonal antibody comprising a heavy chaincomprising the following three CDRs, CDR-H1, CDR-H2 and CDR-H3 of aminoacid sequences SEQ ID N^(o) 16, 17 and 18, respectively, and a lightchain comprising the following three CDRs, CDR-L1, CDR-L2 and CDR-L3 ofamino acid sequences SEQ ID N^(o) 19, 20 and 21, respectively.
 16. Aproduct/computer program containing a set of instructions characteristicof implementation of the method of any one of claims 1 to
 14. 17. Aprocessing system including a computation unit and an input interface,said system including means for implementing the method of any one ofclaims 1 to 14.